Project Summary The long-term goal of my research is to comprehensively characterize the RNA structurome, here defined as the collection of all RNA structures and RNA-RNA interactions in living cells. RNA structures represent an important, yet under-appreciated, layer of genetic information that is essential for the interpretation and execution of the genomic blueprint. In this application for career development award, I have outlined research strategies that range from development of methods for RNA structure determination to functional characterization of RNA structures, which will eventually lead to applications to human diseases, the ultimate goal of genome medicine. RNA structures and interactions are fundamental to RNA's diverse functions, such as guiding, scaffolding and catalysis. Not surprisingly, genetic alternations of RNA structures or helicases (enzymes that remodel RNA structures) underlie many human diseases, including various cancers. RNA viruses cause some of the most deadly human infections, and viral genomic RNA structures control critical steps in their lifecycle. However, only a few RNA structures have been determined due to lack of proper methods. To address this issue, I developed PARIS, a psoralen-crosslinking based method for high throughput mapping of RNA duplexes in living cells at single-molecule level with near base-pair resolution (Lu et al. 2016 Cell). In this proposal, I will further increase the capabilities of PARIS by developing new high-efficiency photochemical crosslinkers (high solubility psoralens and bifunctional carbazoles) and resolution-refining enzymatic strategies. PARIS-determined structures made it possible to conduct global screens for their functions using synthetic translation reporters. Systematic mutagenesis and screening of protein effectors will be used to dissect the mechanism of function for regulatory structural elements. In summary, the proposed studies will deliver a set of powerful tools to the broad RNA community for RNA structure determination, and provide new insights into RNA functions through multi-scale interrogation of RNA structures, from domains to single base pairs. Comprehensive characterization of the RNA structurome will uncover the regulatory mechanisms that translate the genome to phenotype, in both physiological and disease contexts.